Over the last period, significant progress has been made in understanding the nature of the Alz-50 antigen in the Alzheimer brain, and using immunoaffinity chromatography with an IgG1 class switch variant of Alz-50, a new method for isolation of paired helical filaments (PHF) has been developed. Material purified by this technique has been used to raise two new series of monoclonal antibodies, and a more efficient immunoaffinity purification of PHF has been developed. In this completely revised application, we propose to further characterize the purified PHF, using both biochemical, immunoblotting and HPLC techniques, and structural (circular dichroism, Fourier-transformed infrared spectroscopy, X-ray diffraction) analysis, together with electron microscopy (Specific Aim 1). Attempts will be made to define the role of specific polypeptides and functional groups on the assembly of paired helical filaments. A protein kinase activity present in the highly purified PHF preparations will be further characterized in Specific Aim 2, especially in terms of substrate specificity. Further characterization of the new monoclonal antibodies will also be carried out. Two particularly interesting new monoclonal antibodies have been developed over the last project period. TG3 is an IgM which appears to be superior reagent in immunocytochemical studies of formation fixed and paraffin embedded human autopsy tissues. Preliminary work has been unable to demonstrate any reactivity of this antibody with phosphorylated tau, although there is reactivity with paired helical filaments. Under Specific Aim 3, studies of the nature of the TG3 epitope in PHF will be performed. MC16 is an IgG2a which stains nuclei in both the normal and AD brain, but also reacts with tangles and with neuritic elements and amyloid in the AD brain. By immunoblotting, the major reactive protein appears to be a 3kDa protein, and there is no reactivity of this antibody with phosphorylated tau. In Specific Aim 4, this protein will be purified from human brain and sequenced to determine its identity. Given the large number of different PHF-related epitopes which can now be examined, attempts will be made in Specific Aim 5 to use immunoblotting and ELISA techniques to stage the appearance of these epitopes in relation to the development of AD pathology, using the neuropathologic staging system of Braak.